The METTL3-YTHDC1 axis mediates architectural RNA m6A modification to modulate the integrity of chromatin TADs in MLLr+ AML genome

Methyltransferase-like protein 3 (METTL3) as a key regulator of leukemia-associated gene expression in acute myeloid leukemia (AML) contributes to malignant transformation. Chromatin topologically associating domains (TADs) are essential for maintaining the structural integrity and activity of the AML genome. However, the regulatory mechanism by which METTL3 facilitates the integrity and activity of chromatin TADs leading to AML progression remains unknown. Our findings demonstrate that METTL3 has a specific interaction with YTHDC1, METTL3 and CCCTC-binding factor (CTCF) by METTL3 immunoprecipitation-mass spectrometry (IP-MS) analysis in MLLr+ AML cells. METTL3 colocalizes with CTCF at the promoters and enhancers of the MLLr+ AML genome by analysis of METTL3 ChIP-seq and CTCF ChIP-seq. Notably, METTL3 is highly upregulated in MLLr+ AML cells. Depletion of METTL3 significantly impacts CTCT binding sites (CBSs) and chromatin accessibility at the promoters and enhancers of key leukemia genes, such as MYB and RUNX1, resulting in reduced leukemia gene expression and impaired cell proliferation. Critically, high-resolution chromatin conformation capture (Hi-C) data indicates that YTHDC1 loss substantially disrupts CTCF-dependent 3D genome organization. Furthermore, disruption of chromatin architectural RNAs (arcRNAs) mediated R-loops influences the chromatin TAD structures at specific leukemia signature gene loci. YTHDC1 recognizes m6A-modified arcRNAs, enhancing their formation of R-loops to maintain the CTCF-dependent TADs in MLLr+ AML genome. This interaction is crucial for regulating oncogenic TAD boundary activity and integrity to modulate the transcription of leukemia signature genes. In conclusion, the METTL3-YTHDC1-CTCF axis plays a critical role in regulating the expression of AML signature genes through facilitating AML genome organization. These findings provide a valuable theoretical insight for identifying new therapeutic targets in AML treatment.